Previous Article | Next Article 
Journal of Clinical Microbiology, April 1998, p. 1146-1150, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Disseminated Invasive Infection Due to
Metarrhizium anisopliae in an Immunocompromised
Child
David
Burgner,1
Gillian
Eagles,1
Margaret
Burgess,2
Peter
Procopis,3
Maureen
Rogers,4
David
Muir,5
Robert
Pritchard,5,*
Ailsa
Hocking,6 and
Michael
Priest7
Departments of
Microbiology,1
Oncology,2
Neurology,3 and
Dermatology,4 Royal Alexandra Hospital
for Children, Westmead, NSW 2145, Australian National Reference
Laboratory in Medical Mycology, Royal North Shore Hospital, St.
Leonards, NSW 2065,5
CSIRO Division of
Food Science and Technology, North Ryde, NSW
2113,6 and
NSW Agriculture, Plant
Pathology Herbarium, Agricultural and Veterinary Centre, Orange,
NSW 2800,7 Australia
Received 15 July 1997/Returned for modification 19 August
1997/Accepted 9 January 1998
 |
ABSTRACT |
The first reported human case of possible disseminated infection
with the insect pathogen Metarrhizium anisopliae var.
anisopliae, a fungus which has been used commercially for
biocontrol of insects, is described. The patient, a 9-year-old boy, had
a 5-year history of pre-B-cell acute lymphoblastic leukemia and had
been on chemotherapy throughout this period. After 10 days of
profound neutropenia, lesions consistent with ecthyma gangrenosum
appeared on his arms and legs. M. anisopliae was grown from
specimens from three separate sites, collected at different times over
a period of 1 month: a skin biopsy, a swab from the base of a lesion,
and the core of another skin lesion which spontaneously discharged. The
initial skin biopsy also showed histological evidence of epidermal
necrosis and dermal invasion with fungal hyphae. A computed-tomography (CT) scan of the chest demonstrated a lesion in the superior segment of
the lower lobe of the left lung. A CT scan of the brain revealed a
lesion in the left temporoparietal region of the brain, consistent with
an abscess. Despite antifungal treatment including liposomal amphotericin and 5-flucytosine, the patient eventually died. The initial portal of entry is unknown, but hematogenous dissemination to
the skin appears likely because of the multiple ecthymic lesions, and
the appearances of the brain lesion on the CT scan are consistent with
a hematogenous fungal abscess.
| |
TEXT |
Advances in the treatment of many
childhood malignancies have improved prognosis, but intensive
chemotherapy regimens have increased susceptibility to opportunistic
infections, particularly to deep fungal infections (14).
Such infections are increasingly important causes of morbidity and
mortality in neutropenic children (9). The fungi most often
encountered are Candida and Aspergillus (14).
We describe the first reported case of possible disseminated infection
with the insect pathogen Metarrhizium anisopliae
(Metschnikow) Sorokin var. anisopliae. This fungus has been
used commercially for biocontrol of insects.
Case report.
The patient, a 9-year-old boy, had a 5-year
history of pre-B-cell acute lymphoblastic leukemia and had been on
chemotherapy throughout this period. The condition had relapsed twice,
with bone marrow involvement but without central nervous system
involvement. Since the most recent relapse, the boy had been receiving
vincristine, methotrexate (intrathecal), etoposide, asparaginase,
cyclophosphamide, and dexamethasone in a high-dose reinduction
protocol.
He presented, on day 11 of reinduction, with fever to 39°C and
neutropenia (neutrophil count = 0.3 × 109/liter). Blood cultures grew Escherichia coli
and Staphylococcus aureus, and the patient was treated by
the standard oncology neutropenia protocol with
ticarcillin-clavulanic acid (250 mg/kg of body weight/day), vancomycin (40 mg/kg/day), and gentamicin (7 mg/kg/day),
the actual dosages adjusted for renal function as necessary, and
granulocyte colony-stimulating factor. He subsequently became afebrile.
Two weeks after this presentation, and after a period of 10 days of
profound neutropenia (neutrophil count < 0.05 × 109/liter), lesions consistent with ecthyma gangrenosum
appeared on the boy's arms and legs. His neutrophil count at this time was 1.5 × 109/liter, and he was afebrile. Because of
the possibility of fungal infection, amphotericin B and itraconazole
(400 mg/day orally) were added to his treatment regimen and
chemotherapy was ceased.
A skin biopsy specimen from a lesion showed histological evidence of
epidermal necrosis, and dermal invasion with fungal hyphae
was seen in
a periodic acid-Schiff-stained section. No leukemic
infiltration of the
skin was seen. The skin biopsy specimen was
processed for detection of
bacteria, fungi, and mycobacteria.
Acid-fast bacilli were not detected
by microscopy. Slivers of
tissue were inoculated onto Sabouraud's
agar, which was incubated
at 35°C in air for 48 h and
subsequently at room temperature.
Ground tissue was cultured on horse
blood agar (in 5% carbon dioxide
and anaerobically), chocolate agar
(in 5% carbon dioxide), MacConkey
agar, and cooked-meat medium.
It was also inoculated onto a Lowenstein-Jensen
slope incubated
in air at 35°C. Diphtheroids were cultured from
the cooked-meat
medium. No mycobacteria were isolated.
After 7 days, one colony of fungus grew on the original Sabouraud's
agar. No fungi grew on any of the bacteriological media.
Because the
fungus was not readily identifiable, it was sent to
the Australian
National Reference Laboratory in Medical Mycology,
where it was
provisionally identified as
M. anisopliae. Because
of the
unusual nature of the isolate and the fact that it had
not previously
been described as a human pathogen, it was initially
considered a
contaminant, although all processing was performed
in a class II safety
cabinet. However, when the same fungus was
isolated from separate
lesions, a skin swab taken 23 days later
and the tissue core of a
lesion taken 3 days after that, it became
clear that it was an invasive
pathogen. The organism grew from
the skin swab specimen after 6 days in
Sabouraud's broth and from
the tissue core specimen after 3 days on
Sabouraud's agar and
chocolate agar (incubated at 35°C in 5%
CO
2) and in Sabouraud's
broth.
The identification was confirmed by the Division of Food Science and
Technology, Commonwealth Scientific and Industrial Research
Organisation (CSIRO), North Ryde, and the Biological and Chemical
Research Institute of the Department of Agriculture, Sydney, New
South
Wales, Australia, as
M. anisopliae (Metschnikow) Sorokin
var.
anisopliae by the culture methods outlined previously
(
13),
with reference to descriptions contained in references
5 and
16 (Fig.
1). The isolate was inoculated onto
Czapek yeast extract
agar (CYA) and malt extract agar (MEA) and grown
at 25 and 37°C.
Later, its ability to grow at temperatures between 35 and 37°C
was examined in more detail.
View larger version (182K):
[in this window]
[in a new window]
|
FIG. 1.
M. anisopliae. (A) Colonies on CYA (left) and
MEA (right) after 14 days at 25°C; (B) aggregated conidiophores,
showing cylindroidal phialides producing ellipsoidal conidia; (C)
adherent chains of ellipsoidal conidia. Bars = 10 µm.
|
|
The isolate produced long, dry, adherent chains of dark-olive
ellipsoidal conidia (Fig.
1C) from phialides in aggregated
conidiophores
(Fig.
1B). The aggregation of the conidial chains
into adherent
columns is typical of
M. anisopliae
(
5). This isolate compared
well with many reference
cultures held at the DAR Plant Pathology
Herbarium at NSW
Agriculture, Orange, Australia. The fungus grew
well at 25°C
and very weakly at 37°C. This isolate has been deposited
in the
culture collection of the CSIRO Division of Food Science
and
Technology, North Ryde, New South Wales, Australia, as isolate
FRR
4834.
To determine its potential as a pathogen, the isolate was grown at 35, 36, and 37°C. Temperatures were accurate to within
± 0.2°C. After 7 days, growth at 35°C was 12 to 14 mm in diameter
on
CYA and 8 to 10 mm on MEA. At 36°C, growth was 4 to 5 mm (CYA)
and 3 to 4 mm (MEA), and at 37°C, the isolate formed microcolonies
on both
media but growth on CYA was slightly stronger. There was
no sporulation
at 37°C after 7 days.
By in vitro antifungal susceptibility testing, by a tablet
diffusion method (
20), carried out at the Australian
National
Reference Laboratory in Medical Mycology, it was found that
the
organism was resistant to itraconazole, fluconazole, ketoconazole,
and 5-flucytosine but sensitive to amphotericin B.
This organism was grown from specimens from three separate sites
collected at different times over a period of 1 month: the
initial
biopsy, a swab from the base of a lesion, and the core
of another of
the skin lesions which spontaneously discharged.
Within 2 weeks of the
appearance of the first lesions, a total
of 65 lesions of ecthyma
gangrenosum appeared over the patient's
limbs, trunk, and scalp.
Thirty-eight separate blood cultures
(with blood taken from the time of
the appearance of the first
lesion until the time of death, by using
Hemoline diphasic bottles)
did not yield
M. anisopliae. Twenty-eight of the 38 blood cultures
underwent
prolonged incubation for 21 days, to maximize the chance
of recovering
the fungus if it were present.
A chest radiograph taken at the onset of the ecthyma gangrenosum showed
an opacity in the left lung, and a computed-tomography
(CT) scan of the
chest demonstrated this lesion to be in the superior
segment of the
lower lobe. The lesion demonstrated areas of central
lucency consistent
with small areas of cavitation (Fig.
2).
An
abdominal CT scan was normal, and an echocardiogram did not
demonstrate
fungal lesions within the heart or associated with the
central
venous catheter.
View larger version (93K):
[in this window]
[in a new window]
|
FIG. 2.
CT scan of the patient's chest demonstrating a lesion
(arrowhead) with areas consistent with small areas of cavitation in the
superior portion of the lower lobe of the left lung.
|
|
Six days after the appearance of ecthyma gangrenosum, the patient
complained of headache and photophobia. A CT scan of the
brain showed a
wedge-shaped area of low density in the left temporoparietal
region;
this resembled an infarct and had no associated mass effect
or edema.
Over the next week, he became increasingly drowsy and
confused and had
a prolonged generalized seizure. A further brain
CT scan indicated that
the lesion had increased in size and that
there was surrounding edema.
Postictally the patient remained
deeply obtunded. His antifungal
treatment was changed to liposomal
amphotericin, and 5-flucytosine (150 mg/kg/day, adjusted for renal
function) was added. His parents did not
grant permission for
a brain biopsy. A further CT scan of the brain
suggested organization
and probable cavitation of the lesion in the
parietal region,
with surrounding edema, consistent with an abscess
(Fig.
3). The
patient remained comatose
for a month. No new skin lesions appeared
during this period, and
antifungal and antibiotic therapy was
continued.
View larger version (121K):
[in this window]
[in a new window]
|
FIG. 3.
CT scan of the patient's brain demonstrating the lesion
in the left parietal region. The lesion had an appearance consistent
with an abscess, with probable cavitation and surrounding edema.
|
|
The patient's level of consciousness gradually improved, but he had
right hemiparesis and nominal dysphasia. Further antileukemic
chemotherapy was withheld because of continuing fungal sepsis.
Three
months after presentation, the leukemia returned again,
with 50%
lymphoblasts in his peripheral blood. After consultation
with his
parents, in view of the poor prognosis and lack of a
suitable bone
marrow donor, the decision was made not to recommence
further
chemotherapy. The child's peripheral lymphoblast count
continued to rise, and he became neutropenic. The initial ecthyma
lesions never resolved, in spite of the antifungal therapy, and
further
ecthyma gangrenosum lesions appeared on his limbs when
he again became
neutropenic despite continuing treatment with
amphotericin. The
new lesions were not cultured. He died 4 days
later. Permission
for a postmortem examination was refused. Neither
lumbar puncture nor
bronchoscopy was performed at any time.
Discussion.
M. anisopliae was cultured from this
patient from three different cutaneous lesions on three separate
occasions, and clinical, histological, and radiographic findings were
consistent with a disseminated fungal infection. Although the patient
eventually died from his underlying malignancy, the fungal infection
caused severe morbidity and contributed to his death, as disseminated M. anisopliae infection precluded further chemotherapy.
While the initial portal of entry is unknown, hematogenous
dissemination of the organism to the skin and possibly the brain
appears likely.
M. anisopliae is well recognized as an insect pathogen with
a worldwide distribution. It is being used for biological control
of
insects belonging to the orders Coleoptera and Orthoptera,
(beetles
[
21] and locusts [
8]) and is
currently registered
for control of the redheaded pasture cockchafer in
Australia under
the trade name Biogreen. It is also relatively common
in forest
and cultivated soils throughout the world (
5). The
optimum
temperature for growth is 25°C, with a generally recognized
maximum
near 35 to 36°C. In a study of 204 isolates of
M. anisopliae,
Yip et al. (
21) were able to recognize
three groups based upon
their ability to grow at 5 or 37°C. At
37°C, several isolates
produced colonies up to 6 mm in diameter after
7 days. The pH
range for growth is 3.3 to 8.5.
M. anisopliae is capable of decomposing wool and chitin, and
some isolates may be weakly lipolytic and proteolytic (
5).
It has been reported to produce a range of toxic metabolites,
including
cytochalasins C and D, which are highly toxic to mouse
fibroblasts in culture (
5), and destructins (cyclic
peptides),
which have been suggested to suppress cellular immune
response
to the pathogen in insects (
12).
Ecthyma gangrenosum is indicative of disseminated sepsis. The lesions
typically begin as erythematous or purpuric macules
and progress to
form indurated painless necrotic or hemorrhagic
bullae (
2).
The lesions may be single or multiple and typically
occur on the trunk
and the limbs. Histologically, there is invasion
of the dermis with
necrosis of the epidermis and dermis. Invading
organisms may be seen in
the dermal blood vessels, and skin biopsy
culture is often positive
(
2). Ecthyma gangrenosum is described
to occur in infection
with
Pseudomonas aeruginosa (
2,
6),
Stenotrophomonas (
Xanthomonas)
maltophilia (
17), and
Klebsiella pneumoniae (
15). Fungal infection with
Fusarium species (
10)
and
Scytalidium
dimidiatum (
1) has also been associated with
the
development of ecthyma gangrenosum, but as this case illustrates,
other
fungi can cause the characteristic lesions.
While there is no direct evidence that this patient had a
cerebral abscess due to
M. anisopliae, the presence of
multiple
ecthymic skin lesions is strongly suggestive of hematogenous
dissemination,
and the cerebral lesion seen on the CT scan is
consistent with
a fungal abscess. Fungi are common causes of brain
abscesses in
immunocompromised patients, especially in those with
neutropenia
or following marrow transplantation (
7,
19).
Occasional cases
occur in the apparently immunocompetent
(
19).
Aspergillus and
Candida are the
most common causes (
7), although a variety
of other
opportunistic fungi have been described, including
Rhizopus species (
7), phaeohyphomycetes (
7,
11), and,
particularly,
Cryptococcus species (
19). The
outcome is poor, with a very
high mortality rate (
19). To
the best of our knowledge,
M. anisopliae has not previously
been reported as a cause of brain abscess or
invasive disease in
humans. One human isolate was apparently collected
from sputum
and deposited in the Centralbureau voor Schimmelcultures
in 1952 (
3).
M. anisopliae has also recently been
reported
as a cause of keratitis in a patient from Colombia
(
4).
We have no evidence to suggest that the infection in this patient was
connected with commercial usage of
M. anisopliae.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Australian
National Reference Laboratory in Medical Mycology, Royal North Shore
Hospital, Pacific Hwy., St. Leonards, NSW 2065, Australia. Phone:
612-9926-8477. Fax: 612-9437-5746. E-mail:
rcpmicro{at}med.usyd.edu.au.
| |
REFERENCES |
| 1.
|
Benne, C. A.,
C. Neelman,
M. Bruin,
G. S. de Hoog, and A. Fleer.
1993.
Disseminating infection with Scytalidium dimidiatum in a granulocytopenic child.
Eur. J. Clin. Microbiol. Infect. Dis.
12:118-121[Medline].
|
| 2.
|
Blumenthal, N. C.,
U. R. Sood,
P. J. Aronsen, and K. Hashimoto.
1990.
Facial ulcerations in an immunocompromised patient.
Arch. Dermatol.
126:527-532.
|
| 3.
|
Centralbureau voor Schimmelcultures Baarn.
1994.
List of cultures, 33rd ed.
Centralbureau voor Schimmelcultures, Baarn, The Netherlands.
|
| 4.
|
Cepero de Garcia, M. C.,
M. L. Arboleda,
F. Barraquer, and E. Grose.
1997.
Fungal keratitis caused by Metarrhizium anisopliae var. anisopliae.
J. Med. Vet. Mycol.
35:361-363[Medline].
|
| 5.
|
Domsch, K. H.,
W. Gams, and T.-H. Anderson.
1980.
Compendium of soil fungi.
Academic Press, London, United Kingdom.
|
| 6.
|
Fergie, J. E.,
C. C. Patrick, and L. Lott.
1991.
Pseudomonas aeruginosa cellulitis and ecthyma gangrenosum in immunocompromised children.
Pediatr. Infect. Dis. J.
10:496-500[Medline].
|
| 7.
|
Hagensee, M. E.,
J. E. Bauwens,
B. Kjos, and R. A. Bowden.
1994.
Brain abscess following marrow transplantation: experience at the Fred Hutchinson Cancer Research Center, 1984-1992.
Clin. Infect. Dis.
19:402-408[Medline].
|
| 8.
|
Hall, R. A., and B. Papierok.
1982.
Fungi as biological control agents of arthropods of agricultural and medical importance.
Parasitology
84:205-240[Medline].
|
| 9.
|
Koll, B. S., and A. E. Brown.
1993.
Changing patterns of infections in the immunocompromised patient with cancer.
Hematol. Oncol. Clin. North Am.
7:753-769[Medline].
|
| 10.
| Martino, P., R. Gastaldi, R. Raccah, and C. Girmenia. 1994. Clinical patterns of Fusarium
infections in immunocompromised patients. J. Infect.
28(Suppl. 1):7-15.
|
| 11.
|
Palaoglu, S.,
A. Sav,
T. Basak,
Y. Yalcinlar, and B. W. Scheithauer.
1993.
Cerebral phaeohyphomycosis.
Neurosurgery
33:894-897[Medline].
|
| 12.
|
Parry, M.
1995.
A review of mycochemical and insect interactions.
Biocontrol News Inf.
16:27N-33N.
|
| 13.
|
Pitt, J. I., and A. D. Hocking.
1997.
Fungi and food spoilage, 2nd ed.
Blackie Academic and Professional, London, United Kingdom.
|
| 14.
| Pizzo, P. A., and T. J. Walsh. 1990. Fungal infections in the paediatric cancer patient. Semin. Oncol.
17(Suppl 6):6-9.
|
| 15.
|
Stotka, J. L., and M. E. Rupp.
1991.
Klebsiella pneumoniae urinary tract infection complicated by endophthalmitis, perinephric abscess, and ecthyma gangrenosum.
South. Med. J.
84:790-793[Medline].
|
| 16.
|
Tulloch, M.
1976.
The genus Metarhizium.
Trans. Br. Mycol. Soc.
66:407-411.
|
| 17.
|
Vartivarian, S. E.,
K. A. Papadakis,
J. A. Palacios,
J. T. Manning, and E. J. Anaissie.
1994.
Mucocutaneous and soft tissue infections caused by Xanthomonas maltophilia. A new spectrum.
Ann. Intern. Med.
121:969-973[Abstract/Free Full Text].
|
| 18.
|
Wilson, E.
1982.
Cerebral abscess caused by Cladosporium bantianum. Case report.
Pathology
14:91-96[Medline].
|
| 19.
|
Wispelwey, B., and M. W. Scheld.
1995.
Brain abscess, p. 890.
In
G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious disease, 4th ed. Churchill, New York, N.Y.
|
| 20.
|
Wood, G.,
J. McCormack,
D. Muir,
D. Ellis,
M. Ridley,
R. Pritchard, and M. Harrison.
1992.
Clinical features of human infection with Scedosporium inflatum.
Clin. Infect. Dis.
14:1027-1033[Medline].
|
| 21.
|
Yip, H. Y.,
A. C. Rath, and T. B. Koen.
1992.
Characterization of Metarhizium anisopliae isolated from Tasmanian pasture soils and their pathogenicity to redheaded pasture cockchafer (Coleoptera: Scarabaeidae: Adoryphous couloni).
Mycol. Res.
96:92-96.
|
Journal of Clinical Microbiology, April 1998, p. 1146-1150, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Darbro, J. M., Thomas, M. B.
(2009). Spore Persistence and Likelihood of Aeroallergenicity of Entomopathogenic Fungi Used for Mosquito Control. Am J Trop Med Hyg
80: 992-997
[Abstract]
[Full Text]
-
Osorio, S., de la Camara, R., Monteserin, M. C., Granados, R., Ona, F., Rodriguez-Tudela, J. L., Cuenca-Estrella, M.
(2007). Recurrent Disseminated Skin Lesions Due to Metarrhizium anisopliae in an Adult Patient with Acute Myelogenous Leukemia. J. Clin. Microbiol.
45: 651-655
[Abstract]
[Full Text]
-
Revankar, S. G., Sutton, D. A., Sanche, S. E., Rao, J., Zervos, M., Dashti, F., Rinaldi, M. G.
(1999). Metarrhizium anisopliae as a Cause of Sinusitis in Immunocompetent Hosts. J. Clin. Microbiol.
37: 195-198
[Abstract]
[Full Text]
Top
Abstract
Text
